巨细胞病毒对人胚成纤维细胞β肌动蛋白mRNA及微丝的影响

目的：探讨人巨细胞病毒（CMV）对人胚成纤维细胞（HF）的感染及其对细胞肌动蛋白基因 （β-actin）mRNA表达水平和微丝的影响。方法： 应用细胞形态学、RT-PCR半定量法、透射电镜观察CMV对HF细胞的感染及感染后细胞β-actin mRNA和微丝的变化。结果： 经CMV作用后，HF细胞形态由梭形变粗、变圆甚至脱壁；HF细胞有CMV即刻早期抗原基因(IE)mRNA表达，且随着CMV滴度的增加，IE mRNA相对表达量逐渐升高, 而β-actin mRNA相对表达量逐渐减弱，甘油醛3-磷酸脱氢酶基因 （GAPDH） mRNA表达无明显变化。透射电镜下，在HF细胞核、浆内见到大量CMV病毒颗粒；并有细胞微丝断裂、减少，排列紊乱。结论： CMV能感染HF细胞，并在其中活化复制，且致细胞β-actin mRNA表达水平降低；微丝形态、结构发生改变。     

Human cytomegalovirus infection during childhood: detection of viral DNA in peripheral blood by means of polymerase chain reaction

Polymerase chain reaction (PCR) technique was applied to detect cytomegalovirus (CMV) DNA. Two pairs of synthetic oligonucleotide primers were used to amplify DNA from the immediate early 1 and the late antigen genes of CMV, respectively. Either primer sets could detect as few as 0.01 plaque-forming unit of CMV strain AD 169 by Southern blot hybridization. Sixteen CMV clinical isolates were examined and all were found to be positive by the both primer sets. The PCR was used to detect CMV DNA in peripheral blood from six children with elevated anti-CMV antibody titers, who showed abnormal liver-function tests. In three immunocompromised patients, all blood samples were positive for CMV DNA. In three immunocompetent young infants with primary CMV infection, CMV DNA was detected from peripheral blood of one patient during acute phase. Presence of CMV DNA in peripheral blood seemed to be related with the extent of CMV infection, and possibly diagnostic for CMV hepatitis.     

Comparison between viremia and antigenemia for detection of cytomegalovirus in blood.

In a prospective study, 139 serial blood samples from 15 transplant recipients were assessed for the presence of cytomegalovirus (CMV) by virus isolation (CMV viremia) and by direct staining of CMV antigens (CMV Ag) in blood leukocytes (CMV antigenemia). CMV was isolated from 23 samples, whereas CMV Ag was detected in 44 specimens. All positive samples were from a total of nine patients who were diagnosed as having active CMV infections. In seven patients, active CMV infections were diagnosed by virus isolation from blood and urine and by a significant rise of CMV-specific antibodies. In these patients, 21 of the 23 blood samples which were positive for CMV by cell culture were also positive by direct CMV Ag detection. Moreover, CMV Ag were detected in 23 of the 116 culture-negative samples. Twenty of these samples were from the acute phase of infection in the same seven patients. The remaining three CMV Ag-positive specimens were from the other two patients, from whom CMV was not isolated but who had serological evidence of concomitant active CMV infections. These results suggest that direct detection of CMV Ag in peripheral blood leukocytes is as specific as and more sensitive than current isolation techniques. Furthermore, by its sensitivity and inherent rapidity the antigen detection test proved to be the earliest diagnostic marker of active CMV infection in eight of the nine patients. Finally, it was shown that monoclonal antibodies to CMV immediate early antigens are a prerequisite for demonstration of CMV antigenemia.     

Cytomegalovirus diagnosis in renal and bone marrow transplant recipients: the impact of molecular assays.

BACKGROUND: Cytomegalovirus (CMV) infections are a major threat in transplant recipients. In recent years, new assays for routine CMV diagnosis, based on molecular techniques, have become available. OBJECTIVE: The impact of molecular assays for CMV diagnosis in transplant recipient was evaluated. STUDY DESIGN: A total of 51 transplant recipients were screened for CMV infection. Serological (AxSYM CMV IgG and recombinant CMV IgM assays), antigenemia, CMV DNA (qualitative in house PCR and the quantitative COBAS AMPLICOR CMV MONITOR Test), and CMV mRNA (NucliSens CMV pp67 Test) tests were compared. RESULTS: In 11/20 bone marrow transplant (BMT) recipients and 10/31 renal transplant (RTX) recipients there was no evidence of active CMV infection. Ten RTX recipients and one BMT recipient were antigenemia positive, 21 RTX and seven BMT recipients were PCR positive (qualitative CMV PCR). There were more BMT recipients CMV DNA positive in serum (7/21) than antigenemia positive (1/21). CMV mRNA was found positive in two BMT recipients (one case with no other evidence of CMV infection, the other one CMV DNA positive and antigenemia negative). The only antigenemia positive BMT recipient was found negative for CMV mRNA, but positive in all other tests. Eight RTX recipients were found positive for CMV mRNA. Six of them were also antigenemia positive and five of those were also found positive for CMV IgM. One CMV mRNA positive RTX recipient was CMV IgM positive but antigenemia negative and the other one CMV mRNA positive RTX recipient was found negative in all other tests. Two antigenemia positive RTX recipients were found negative for mRNA and CMV IgM. CONCLUSION: Antigenemia was found to be a good screening test for CMV infection in RTX recipients. In BMT recipients, tests based on molecular techniques appeared to be superior compared to antigenemia.     

Detection of cytomegalovirus infection during a vaccine clinical trial in healthy young women: seroconversion and viral shedding.

BACKGROUND: An antibody method based on absorption of serum with cytomegalovirus (CMV) glycoprotein B (gB) was developed for detection of infection during clinical trials of CMV gB vaccine. Previous study showed that this method detected the antibody response to infection and was negative with vaccine induced immunity. OBJECTIVES: In an ongoing efficacy trial of CMV gB vaccine the ability of the gB-absorbed CMV IgG assay to detect CMV infection was assessed and compared with viral culture results. STUDY DESIGN: Two hundred and ninety two healthy, seronegative young women in a phase II, double-blind, placebo-controlled, clinical trial of recombinant CMV gB vaccine (sanofi pasteur) with MF59 adjuvant (Chiron) were randomized to receive CMV gB vaccine or placebo (1:1) on a 0, 1 and 6 month schedule. Participants were screened every 3 months for CMV infection using the gB-absorbed CMV IgG assay, and a subgroup was also screened for infection with viral cultures. Viral culture (urine, vaginal swab and saliva) was used to confirm CMV infection in all subjects with a positive gB-absorbed CMV IgG result. RESULTS: Evidence of CMV infection (gB-absorbed CMV IgG levels>or=5.0 AU/ml) was found in 23/292 (7.88%) study participants. The gB-absorbed CMV IgG levels of their first positive serum ranged from 15.7 to 251.0 AU/ml with a mean of 77.0 AU/ml and a median of 44.9 AU/ml. Cytomegalovirus was isolated from all 23 of them from culture specimens collected after their first positive gB-absorbed CMV IgG. The time to first CMV positive culture from first positive gB-absorbed CMV IgG ranged from 0 to 12 weeks with a median of 2 weeks. CONCLUSIONS: The gB-absorbed CMV IgG assay detects CMV infection in CMV gB vaccine clinical trials earlier and more rapidly than virus culture and does not reveal whether subjects received CMV gB vaccine or placebo.     

Cytomegalovirus infection in heart transplant recipients: Preliminary results of a controlled trial of intravenous gamma globulin

Cytomegalovirus (CMV) infection is a major problem during the first twelve weeks after cardiac transplantation. Reactivation of endogenous virus, blood transfusion, and the transplanted heart are sources for CMV infection. Not only can disseminated CMV infection contribute directly to mortality, but CMV infection predisposes to superinfection with bacterial and fungal pathogens. Studies in mice, the premature neonate, and bone marrow transplant recipients suggest that passive immunization with CMV antibody has a modifying effect on CMV infection. By prophylactic administration of intravenous immune globulin in a controlled randomized fashion to cardiac transplant recipients we will address the question of whether passively acquired antibody prevents or modifies infection with CMV or other viral, bacterial, and fungal infections. Thirteen patients undergoing cardiac transplantation at Stanford University were randomized to receive or not receive intravenous immune globulin (IGIV) at a dose of 20 ml/kg twenty-four hours post transplantation and then weekly thereafter for a period of 10 weeks. Six patients seronegative for CMV antibody were randomized separately from seven who were seropositive for CMV antibody. Seven patients randomly selected from these two groups received IGIV. In patients seronegative for CMV, administration of IGIV resulted in serum titers of 1:10,000–1:30,000 measured by radioimmunoassay. Antibody titers were maintained at levels of 1:10,000 to 1:30,000 throughout the 10 week period post transplantation. Antibody remained detectable up to 8 weeks after the last infusion. No significant increases in CMV antibody levels were detectable after IGIV administration in patients seropositive for CMV antibody prior to transplantation. The administration of IGIV in thirty-two large and repeated doses was safe and well tolerated in cardiac transplant recipients. Serum levels of CMV antibody comparable to those observed in normal CMV seropositive individuals were achieved in CMV seronegative transplant recipients and were maintained throughout the period of greatest risk for CMV infection.     

Detection of cytomegalovirus DNA in sera of liver transplant recipients.

We prospectively studied the utility of the amplification of cytomegalovirus (CMV) DNA in the sera of liver transplant recipients in order to predict symptomatic CMV infection, thus enabling preemptive therapy with antiviral agents. Serum samples obtained at biweekly intervals from 20 sequential liver transplant recipients for at least 8 weeks following transplantation were tested by the PCR amplification procedure. Results were correlated with blood and urine cultures, histopathological findings from infected organs, and clinical manifestations. Six patients (30%) developed symptomatic CMV infection; in five (83%) of these patients, CMV DNA was detected prior to symptomatic CMV infection, and in one (17%) of these patients, CMV DNA was detected at the time of symptomatic CMV infection. CMV DNA was detected a mean of 13 days (range, 0 to 23 days) prior to the onset of symptomatic CMV infection. In addition, CMV DNA was detected in the sera of four of five patients with asymptomatic viremia and two patients with asymptomatic viruria. Lastly, the PCR was negative for sera from seven patients with no evidence of CMV infection. We found that PCR was able to detect the presence of CMV DNA in the sera of liver transplant recipients at a sensitivity of 92% and a specificity of 100% for CMV infection, while the sensitivity and specificity for symptomatic infection were 100 and 57%, respectively.     

Evaluation of the Abbott AxSYM Cytomegalovirus (CMV) Immunoglobulin M (IgM) Assay in Conjunction with Other CMV IgM Tests and a CMV IgG Avidity Assay

The measurement of the avidity of cytomegalovirus (CMV) immunoglobulin G (IgG) antibodies has been shown by several investigators to be useful in identifying and excluding primary CMV infections in pregnant women. In this work, we examined the diagnostic utility of reflex testing of CMV IgM-positive specimens from pregnant women by using a CMV IgG avidity assay. The utility of this approach was directly dependent on the sensitivity of the CMV IgM assay employed during the initial screen. The higher initial reactivity rate of the AxSYM CMV IgM assay was necessary in order to detect CMV IgM in specimens containing low-avidity CMV IgG antibodies, indicative of a primary CMV infection, which other CMV IgM assays (Behring, Vidas, Captia, and Eurogenetics) fail to detect in some cases. The use of the AxSYM CMV IgM assay, followed by an avidity test, should result in more accurate diagnosis of CMV infection in pregnant women.     

Usefulness of pp65 Antigenemia for the Early Diagnosis of Cytomegalovirus Disease in Patients with AIDS

 An observational cohort study was performed to assess the effectiveness of a cytomegalovirus antigenemia (CMV-Ag) assay designed to predict clinical CMV disease in patients with AIDS. Eighty-six HIV-infected patients with CD4+ cell counts of <100/mm³, positive CMV IgG, and no previous CMV disease were enrolled. Thirty-eight (44%) patients had at least one positive CMV antigenemia test, ten of whom eventually developed CMV focal disease. CMV disease was diagnosed in 13 (15%) patients. The CMV antigenemia assay was positive in ten of these 13 patients. Using a cut-off value of five positive cells in every 150 000 leukocytes sampled, the CMV antigenemia assay had a positive predictive value of 89% and a negative predictive value of 94%. The median time from the first positive CMV antigenemia test to the onset of CMV disease was 102 days. CMV disease probability at 6 months in patients with a CMV antigenemia value ≥5 was 77.8% versus 6% in patients with CMV antigenemia value <5 (log-rank test=48.345;P<0.001). Several independent factors were associated with the development of CMV disease: CMV antigenemia ≥5 cells (hazard ratio: 20.44), CD4+ count≤25/mm³ (HR: 3.12), and sexual transmission of HIV infection (hazard ratio, 3.15). CMV antigenemia seems to be a good predictor of CMV disease in patients with AIDS.     

Comparison of In Vitro Lymphocyte Proliferations Induced by Cytomegalovirus-Infected Human Fibroblasts and Cell-Free Cytomegalovirus

In vitro lymphocyte reactivity (LR) to cytomegalovirus (CMV)-infected human fetal fibroblasts (CMVFF) and cell-free CMV were measured by using lymphocytes from healthy donors. Lymphocytes from all seropositive donors were stimulated by CMVFF, whereas lymphocytes from negative donors were not. The optimal stimulator cell-to-lymphocyte ratio was in the range of 1:5 to 1:50, dependent on the virus dose used. LR to cell-free CMV was positive for 15 out of 18 seropositive donors and negative for 14 out of 16 seronegative donors. In most cases LR to CMVFF was considerably higher than LR to cell-free CMV. Within the CMV seropositive group there was no significant correlation between the LR to either CMVFF or cell-free CMV and the levels of antibodies to CMV early antigens or CMV late antigens. There was no strict correlation between LR to CMVFF and to cell-free CMV, especially not in tests with lymphocytes from two patients with CMV mononucleosis. Our data suggest that CMVFF and cell-free CMV are recognized (partly) by different subpopulations of CMV-specific memory lymphocytes. We conclude that the use of CMV-infected cells, in addition to cell-free CMV, in LR tests gives more reproducible and possibly also additional information about CMV-specific cellular immunity.     

Screening of blood donors for cytomegalovirus (CMV) antibodies: an evaluation of different tests

The incidence of transfusion-associated cytomegalovirus (CMV) infections is related to the number of donors and the volume of blood. In immunosuppressed patients primary CMV infection is associated with a high morbidity and/or mortality. These infections can be prevented by the use of CMV negative blood products. A test for screening of CMV antibodies should have a high sensitivity and specificity and in addition be rapid and easily automated. In the present study the routine method in the laboratory, a CMV ELISA using a nuclear CMV antigen, was compared with Vironostika CMV anti-IgG MicroELISA, CMVSCAN, a latex agglutination test and a new ELISA from Organon Teknika, Vironostika CMV one-step MicroELISA. Sera from 419 blood donors were tested by all four tests. Discordant results were resolved by Western blot analysis using a glycine-extracted CMV antigen. The sensitivity was 100% for CMV ELISA and CMVSCAN and 99 and 96% for the CMV anti-IgG and CMV one-step tests respectively. The specificity was 100, 99, 94 and 100%, respectively. The ELISAs are suitable for screening of large numbers of serum samples and the CMV one-step test in particular was rapid and easy to perform although the sensitivity has to be increased. The latex agglutination test has the advantage of being extremely rapid.     

Acquisition of cytomegalovirus infection: an update.

Human cytomegalovirus (CMV) is a ubiquitous deoxyribonucleic acid virus that commonly infects a majority of individuals at some time during their life. Although most of these CMV infections are asymptomatic, certain patient groups are at risk to develop serious illness. Understanding the epidemiology of this virus is a key element in the development of strategies for preventing CMV disease. However, a number of features of this virus complicate such understanding. Following infection, CMV can remain latent, with subsequent reactivation; the factors controlling latency and reactivation and those factors which determine whether a CMV infection will be symptomatic are unknown. CMV disease can be acquired by natural routes, including horizontal and vertical transmission. Due to the ubiquity of CMV, the delineation of CMV transmission by these natural routes is complicated by the myriad of possible sources. Moreover, concerns over the risk of CMV transmission to the seronegative pregnant female have been raised in relation to preventing CMV transmission. By using molecular biologic techniques, much knowledge has been gained regarding the transmission of CMV disease by natural routes; however, a number of questions remain unanswered. The transmission of CMV infection by natural routes is therefore reviewed and the issues are highlighted. Primary infection, reactivation, and reinfection are the types of active CMV infections that can occur in an immunocompromised patient. In addition to natural routes of infection, introduction of presumably latently infected organs and requirements for multiple blood transfusions increase potential exposure to CMV in the immunocompromised patient. Understanding the epidemiology of CMV infections in the immunocompromised patient is difficult and in some instances controversial due to the complexity and interdependency of a number of factors which lead to CMV infection. In an immunocompromised individual, a major risk factor in developing overt CMV-related disease is associated with the serological status of an organ donor, the recipient, and the blood product given to these patients. In addition, a large body of inferential data supports the transmission of CMV by blood products or organs from seropositive donors; however, the mechanisms by which transmission occurs remain unclear. The possible sources and mechanisms of transmission of CMV infections in the immunocompromised host are reviewed. Lastly, strategies for the ultimate prevention of CMV disease are discussed in light of the epidemiology of CMV infections. To date, these strategies have included use of CMV-seronegative blood products or organs, antiviral agents, and vaccines.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of Cytomegalovirus (CMV) IgG Avidity Testing in Diagnosing Primary CMV Infection during Pregnancy

The risk of intrauterine transmission of cytomegalovirus (CMV) during pregnancy is much greater for women who contract primary CMV infection after conception than for women with evidence of infection (circulating CMV antibodies) before conception. Thus, laboratory tests that aid in the identification of recent primary CMV infection are important tools for managing the care of pregnant women suspected of having been exposed to CMV. CMV IgM detection is a sensitive marker of primary CMV infection, but its specificity is poor because CMV IgM is also produced during viral reactivation and persists following primary infection in some individuals. Studies conducted over the last 20 years convincingly demonstrate that measurement of CMV IgG avidity is both a sensitive and a specific method for identifying pregnant women with recent primary CMV infection and thus at increased risk for vertical CMV transmission. IgG avidity is defined as the strength with which IgG binds to antigenic epitopes expressed by a given protein; it matures gradually during the 6 months following primary infection. Low CMV IgG avidity is an accurate indicator of primary infection within the preceding 3 to 4 months, whereas high avidity excludes primary infection within the preceding 3 months. In this minireview, we summarize published data demonstrating the clinical utility of CMV IgG avidity results for estimating time since primary infection in pregnant women, describe commercially available CMV IgG avidity assays, and discuss some of the issues and controversies surrounding CMV IgG avidity testing during pregnancy.     

Conditions for Cytomegalovirus Stimulation of Lymphocytes

Lymphocytes from healthy donors or from patients with chronic lymphocytic leukaemia were subjected to live or inactivated cytomegalovirus (CMV) or the mitogen phytohaemagglutinin. No early or late CMV antigens could be demonstrated in the lymphocytes, indicating that neither abortive nor replicative CMV infection takes place. Only cells from CMV antibody-positive leukaemic and non-leukaemic donors were stimulated by CMV to DNA synthesis, with a maximum on day 5. Cells from all individuals responded to phytohaemagglutinin stimulation, the peak of activity occurring on day 3. The stimulation with CMV occurred in T cells and was independent of early CMV antigen production, viral DNA synthesis, or viral replication. CMV is thus not an in vitro lymphocyte mitogen like Epstein-Barr virus but is a very potent antigen for memory T cells.     

Comparison of PCR, antigenemia assay, and rapid blood culture for detection and prevention of cytomegalovirus disease after lung transplantation

The goal of this study was to evaluate serial cytomegalovirus (CMV) blood culture, antigenemia testing, and qualitative and quantitative plasma CMV PCR for their ability to predict CMV disease and thus to direct preemptive therapy after lung transplantation. Forty-one patients provided 414 samples for blood culture, 290 samples for antigenemia testing, and 432 samples for PCR. Seven patients developed 11 episodes of CMV disease. CMV PCR had sensitivity, specificity, and positive predictive and negative predictive values of 79, 99, 84, and 99%, respectively, compared with 48, 99, 85, and 98%, respectively, for antigenemia testing, and 8, 100, 100, and 97%, respectively, for culture. Only quantitative CMV PCR correlated with disease stage: asymptomatic patients had a mean of 1,500 CMV DNA copies/ml, whereas patients who developed CMV disease had 5,087 copies/ml 12 to 4 weeks before symptoms and 32,000 copies/ml at diagnosis. Furthermore, CMV PCR-measured DNA increased 5- to 10-fold immediately preceding symptoms. PCR and antigenemia test values decreased with anti-CMV therapy. CMV DNA (as detected by PCR), but not antigenemia, persisted in patients who later developed recurrent CMV disease. The data indicate that lung transplant recipients will benefit from monitoring of CMV disease by plasma CMV PCR.     

Combination of native and recombinant cytomegalovirus antigens in a new ELISA for detection of CMV-specific antibodies

BACKGROUND: Serological detection of cytomegalovirus (CMV)-specific antibodies varies greatly due to antigen composition and the lack of antigen standardization. OBJECTIVES: To develop and evaluate a new ELISA with native and/or recombinant cytomegalovirus antigens for the detection of anti-CMV IgG and IgM antibodies. RESULTS: The diagnostic performance of three anti-CMV ELISAs coated with different CMV antigen preparations, (i) native CMV antigen, (ii) a mixture of recombinant CMV peptides pp150, pp28, gB2 and pp52 and (iii) a combination of native CMV antigens and recombinant CMV IE1 antigen applied in the new Genzyme Virotech CMV ELISA, were compared. All tested sera were derived from patients or healthy blood donors and were predefined with the Dade Behring Enzygnost((R)) CMV ELISA as well as by CMV PCR analysis. Additionally, official well-characterized serum panels were also tested. The new Genzyme Virotech CMV ELISA IgG/IgM test applying a combination of native antigens and recombinant IE1 antigen was evaluated and the performance was compared to the Dade Behring Enzygnost((R)) CMV ELISA. The sensitivities were 98.9% (IgG) and 98.2% (IgM), the specificities were 98.8% (IgG) and 98.9% (IgM) for the Genzyme Virotech CMV ELISA. Furthermore all sera of the BBI mixed titer performance panel as well as the BBI seroconversion panel were identified 100% correctly with the new Genzyme Virotech ELISA. CONCLUSIONS: These data suggest that the new Genzyme Virotech CMV ELISA has higher sensitivity and specificity than ELISAs based on native antigens or recombinant peptides only. Specific combinations of native and recombinant antigens increase the serological detection of CMV infections and may add to further standardization of CMV serology.     

Maintenance of cytomegalovirus (CMV) latency and host immune responses of long term renal allograft survivors. I. Prolonged suppression of in vitro lymphocyte responses against CMV infected fibroblasts related to previous secondary CMV infection.

Cytomegalovirus (CMV) specific humoral and cellular immunity was investigated in 16 renal allograft recipients with long term graft survival (26-122 months) who were shown to be CMV seropositive before transplantation. Results were compared with healthy individuals with latent CMV infections. Recipients (n = 9) who experienced a symptomatic secondary CMV infection shortly after transplantation (less than 6 months), showed a prolonged but finally temporary suppression of their in vitro lymphocyte memory responses against CMV infected fibroblasts (CMV-FF; median SI: 1.9), a persistence of high antibody titres against intracellular CMV antigens and most of them also had antibodies against CMV membrane antigens (CMV MA). In contrast the recipients (n = 7) who could maintain their CMV in latency after transplantation, had lower antibody titres and their in vitro memory lymphocyte responses against CMV-FF (median SI: 9.3) were comparable to those of the healthy controls (median SI: 11.6). The memory lymphocyte responses against purified CMV virions were depressed in both recipient groups. These results suggest that cellular immunity against CMV infected target cells constitute an important mechanism in maintaining CMV in latency after allografting.     

Management of allogeneic bone marrow transplant recipients at risk for cytomegalovirus disease using a surveillance bronchoscopy and prolonged pre-emptive ganciclovir therapy.

BACKGROUND: Patients undergoing allogeneic bone marrow transplant (BMT) are considered to be at increased risk of cytomegalovirus (CMV) disease if they and/or their donor are CMV seropositive pre-transplant. Although several pre-emptive strategies have been shown to be effective in preventing early CMV disease, the ability of pre-emptive strategies using prolonged ganciclovir therapy to reduce the incidence of late-onset CMV infection, disease and mortality has not been fully evaluated. OBJECTIVE: To assess the efficacy of 18 weeks of pre-emptive ganciclovir therapy in preventing late-onset (> 100 days post-transplant) CMV disease when administered to asymptomatic BMT patients found to have CMV in bronchoalveolar lavage (BAL) fluid obtained during a surveillance bronchoscopy approximately 35 days post-transplant. To determine whether or not survival of BMT recipients is influenced by pre-transplant donor and recipient CMV serostatus in the context of this pre-emptive ganciclovir strategy. STUDY DESIGN: Consecutive patients undergoing allogeneic BMT were assessed for their risk of developing CMV disease based on their pre-transplant CMV serostatus and that of their donor. Patients who were CMV seropositive and/or received marrow from a CMV seropositive donor underwent a surveillance bronchoscopy and BAL approximately 35 days post-transplant. Patients with positive BAL fluid for CMV received pre-emptive ganciclovir therapy for 18 weeks at decreasing dose levels. Patients considered to be at low risk for the development of CMV disease (donor and recipient CMV seronegative) were followed without intervention. RESULTS: Of 98 consecutive patients, 55 were considered to be at risk for CMV disease and underwent a surveillance bronchoscopy. Sixteen (29%) patients had a positive BAL fluid for CMV and were started on pre-emptive ganciclovir therapy. Two patients progressed and died with CMV-related pneumonia. One additional patient developed CMV-related enteritis on day 42 post-transplant and recovered with continuing ganciclovir treatment. Of the 39 patients with a negative BAL fluid for CMV, one developed a fatal CMV pneumonia 150 days post-transplant and two additional patients developed gastrointestinal CMV disease 28 and 57 days post-BMT, respectively. None of the patients in the low risk group developed CMV disease. CONCLUSIONS: The strategy utilizing a surveillance bronchoscopy for CMV and initiating prolonged (18 weeks) pre-emptive ganciclovir therapy for patients with a positive BAL fluid for CMV resulted in a low incidence of CMV-related post-transplant complications. After a minimum follow-up of 16 months, late CMV reactivations (occurring > 100 days post-transplant) were not observed in the group of individuals pre-emptively treated with ganciclovir. This observation suggests that prolonged therapy with a reduced dose of ganciclovir may be important in the prevention of CMV reactivation. The CMV serostatus of donors and recipients prior to BMT did not correlate with survival.     

Quantitation of cytomegalovirus DNA in lung tissue of bone marrow transplant recipients.

Five bone marrow transplant recipients who died of respiratory failure were retrospectively analyzed with polymerase chain reaction (PCR) assay for pulmonary cytomegalovirus (CMV) infection. Two patients had CMV interstitial pneumonitis according to the virus isolation and the histologic and immunofluorescent examinations of the lungs, while the other three patients had non-CMV diseases (ie, idiopathic interstitial pneumonitis, pulmonary aspergillosis, or Streptococcus mitis septicemia). Cytomegalovirus DNA was amplified from the postmortem lung tissue with PCR. The PCR assay showed apparent PCR signals specific to CMV DNA in the two patients with CMV pneumonitis. In contrast, CMV DNA was hardly detectable or undetectable in the three patients without CMV disease. With quantitative PCR assay the initial CMV copy number in the lung tissue of the two patients with CMV pneumonitis was more than 10(4) copies/micrograms DNA and was over 1,000-fold more than that of the three patients without CMV pneumonitis. These results show that quantitative PCR assay could be useful as a diagnostic measure for pulmonary CMV infection.